Interaction between the cellular prion (PrPC) and the 2P domain K+ channel TREK-1 protein.

The cellular prion protein (PrP(C)) is a highly conserved protein throughout the evolution of mammals and therefore is thought to play important cellular functions. Despite decades of intensive researches, the physiological function of PrP(C) remains enigmatic. Differently, in particular pathological contexts, generally referred as transmissible spongiform encephalopathies, a conformational isoform of PrP(C), i.e., PrP(Sc), is considered the causative agent of these diseases. In this study, we investigated putative PrP(C) cellular functions through the identification of PrP(C) protein interactants. Using a bacterial two-hybrid approach, we identified a novel interaction between PrP(C) and a two-pore potassium channel protein, TREK-1. This interaction was further verified in transfected eukaryotic cells using co-immunoprecipitation and confocal microscopic analysis of the fluorescent transfected proteins. Importantly, in the cerebellar cortex, the endogenous PrP(C) and TREK-1 proteins exhibited co-localization signals in correspondence of the Purkinje cells. Furthermore, a deletion mapping study defined the carboxyl-terminal regions of the two proteins as the possible determinants of the PrP(C)-TREK-1 interaction. Our results indicated a novel PrP(C) interacting protein and suggested that this complex might be relevant in modulating a variety of electrophysiological-dependent cellular responses.

Proliferation and migration of granule cells in the developing rat cerebellum: cisplatin effects.

We evaluated the relationship among proliferation, death and migration of granule cells in lobules VI-VIII of vermis, in comparison with lobule III, during cerebellar development. To this aim, a single injection of cisplatin, i.e., a cytostatic agent that is known to induce death of proliferating granule cells, was given to 10-day-old rats. Histochemical markers of proliferating (PCNA immunoreaction) and apoptotic (TUNEL staining) cells were used; the variations of the external granular layer (EGL) thickness were evaluated in parallel. After PCNA and TUNEL reactions, evident changes of the whole EGL were found on PD11 (1 day after treatment), when a reduction of the thickness of this layer was found in treated rats, mainly in consequence of the high number of apoptotic cells in all the cerebellar lobules. On PD17 (7 days after treatment), a thick layer of proliferating cells was observed in lobules VI-VIII of treated rats, while the peculiar pattern of the normal development showed a thin EGL. At the same time, in treated rats, the number of apoptotic cells in EGL was low. In all developmental stages of treated rats, after GFAP immunoreaction, glial fibers appeared twisted, thickened, and with an irregular course; intensely labeled end-feet were present. The damage of radial glia suggests an alteration of migratory processes of granule cells, which is also evidenced by the decreased thickness of the premigratory zone of the EGL. Injured radial glia fibers were restricted to lobules VI-VIII and they persisted at PD30, leading to the presence of ectopic granule cells in the molecular layer, as we previously described.

Signal molecules and receptors in the differential development of cerebellum lobules. Acute effects of cisplatin on nitric oxide and glutamate systems in Purkinje cell population.

Three functionally correlated parameters, nitric oxide (NO), glutamate and NMDA receptors were analyzed through enzymehistochemical and immunohistochemical reactions. A single injection of cisplatin (cisPt) was administered to 10-day-old rats in order to study how Purkinje cells differentiation may be early changed by a mild injury due to the drug during postnatal cerebellar histogenesis. In comparison with age-matched control rats, a correlated decreasing expression of nitric oxide synthase (NOS), glutamate and NMDAR1 was observed in the Purkinje cells of lobules VI-VIII 6 h after the treatment. Moreover, at 24 h after cisPt, the expression of glutamate, NMDAR1 and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) reactivity was further decreased. In the same period, the ionotropic receptor GluR2 evidenced a less developed dendrite of Purkinje neurons in the top of lobules. In addition, the metabotropic receptor mGluR1alpha revealed unstained areas in the molecular layer, which was entirely stained in control rats; on PD11 this altered pattern was observed in all the lobules and in both the outer and the inner parts. Findings show the importance of NO-glutamate interactions via NMDAR1 in the crucial phases of Purkinje cells differentiation and their involvement on Purkinje neurons dendrite branching as demonstrated by the patterns of the other glutamate receptors. Changes were discussed in relation to an important critical event of Purkinje cell differentiation, i.e. regression of perisomatic spines and elimination of climbing fiber synapses on the somata. Finally, lobules VI-VIII appear to be the most vulnerable ones when cisplatin treatment is administered at 10 days of life, which demonstrates that at this stage some critical developmental changes occur in these lobules and that slower/damaged dendritic tree development is different in the outer versus the inner regions of the lobules.

Nitric oxide synthase in the frog cerebellum: response of Purkinje neurons to unilateral eighth nerve transection.

When vestibular damage occurs, nitric oxide synthase (NOS) expression in rat cerebellar flocculus is affected. Since compensation for postural symptoms occurs and Purkinje cells play an important role in movement coordination and motor learning, we analyzed in situ the induction of NOS in the Purkinje cell population of the cerebellum (corpus cerebelli) of frog after unilateral transection of the eighth statoacoustic nerve to gain insight into the role of NO in neural plasticity after injury. Three days after neurectomy, the early effects induced NADPH diaphorase reactivity in most of the Purkinje cells on the ipsilateral side, while on the contralateral side the highest labeling was observed at 15 days. This finding can give information on the dynamics of vestibular compensation, in which NOS involvement was investigated. At 30 days, NADPH diaphorase reactivity was present in a large number of Purkinje cells of the whole cerebellum, while at 60 days a down-regulation for NADPH diaphorase reactivity was evident. A similar trend was observed for NOS-immunoreactivity, which was still present at 60 days in a high percentage of Purkinje cells, mainly on the ipsilateral side. On the basis of cell density evaluations, it was proposed that the early induction of NOS after neurectomy was linked to the degeneration of a part of the Purkinje neurons, while the permanence of NOS labeling might be due to a neuroprotective role of NO in the restoration phase of the vestibular compensation process.

Cell death may regulate visual functionality in the retina of adults of the dipteran Ceratitis capitata.

The white eye mutation in the medfly Ceratitis capitata, like the homologous mutation in Drosophila melanogaster, was shown to impair visual function. Light and electron microscopy, combined with the DNA-end labelling histochemistry (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) technique), were used to investigate whether programmed cell death may contribute to the morpho-functional differences between the retina of adults from the white eye and wild type strains. Several photoreceptor nuclei in mature white eye flies appeared smaller and showed intensely Toluidine Blue-stained chromatin masses. At the ultrastructural level, they showed different stages of degeneration, resembling apoptotic figures. Positive TUNEL labelling in the white eye retina indicates that apoptosis may be a candidate mechanism for retinal cell degeneration in adult flies, where visual functionality is altered, to achieve the proper cell number. Apoptosis also appears to occur in the wild type retina in early adult life during normal tissue development.